The longterm objective of this grant is to combine morphology, biochemistry, and molecular biology to determine the functional and structural relationships between the basement membrane, epithelial cytoskeleton, and epithelial metabolism. This work will be done on a corneal epithelial cell model which can be used to study the biology of the basement membrane zone by these techniques. The basement membrane (basal lamina) is defined here as consisting of a central dense sheet (lamina densa) rich in laminin and type IV collagen surrounded on both sides by laminae rarae containing glycoproteins and most of the proteoglycan of the basement membrane. The epithelia cell model will be used to determine basement membrane-cytoskeleton relationships by disrupting the cytoskeleton with specific drugs in the presence or absence of an intact basement membrane or added extracellular matrix (ECM) molecules. Is the cytoskeleton necessary for the increase in epithelial collagen production caused by basal cell contact with basement membrane molecules? The second portion of the specific aims is directed toward using probes for the mRNA of collagen and other proteins to determine 1) the location of the mRNA, 2) to make a correlation of mRNA levels with protein synthesis, and 3) the type of protein synthesis regulation (transcription or translation) occurring in these epithelial cells. This proposal does not address a disease process such as Epidermolysis Bullosa directly. However, it will be relevant to understanding the relationship between the ECM, cytoskeleton, and protein synthesis of another ectodermally derived epithelial cell which produces a basement membrane. The results of this proposal will also advance our understanding of corneal epithelial metabolism and differentiation and thus are relevant to eye diseases. We expect these studies to cast light on cell-basement membrane interaction and to open up new pathways for approaching possible effects of abnormal basement membranes on cell metabolism and differentiation.